Ubiquitin Is Conjugated to the Cytoskeletal Protein α-Spectrin in Mature Erythrocytes

Corsi, Dario; Galluzzi, Luca; Crinelli, Rita; Magnani, Mauro

doi:10.1074/jbc.270.15.8928

Cited by 32 publications

(33 citation statements)

References 36 publications

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“…Peptide mapping and Edman degradation analysis showed that biotins were at Lys 6 , Lys 48 , and Lys 63 in tribiotinylated ubiquitin (Table I). Taken together, these data indicate that the order of susceptibility of amino groups in ubiquitin to modification by sulfo-NHS-biotin is Lys 6 Ͼ Ͼ Lys 63 Ͼ Lys 48 Ͼ Ͼ Lys 33 Ͼ Ͼ Lys 11 , Lys 27 , and Lys 29 .…”

Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 74%

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Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Shang

Deng

Liu

et al. 2005

Journal of Biological Chemistry

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Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys 6 of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys 6 was the most readily labeled with sulfosuccinimidobiotin. Lys 6 -biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys 6 -biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys 6 -biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys 6 had similar phenotypes as Lys 6 -biotinylated ubiquitin. Lys 6 mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys 6 -modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys 6 -modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

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Section: Lys 6 Of Ubiquitin Is the Most Readily Labeled With Sulfo-nhmentioning

confidence: 74%

“…Biotinylated ubiquitin has been used as a probe to study the function of the UPP (27). However, biotinylated ubiquitin has not been characterized in detail, and the potential kinetic artifacts of biotinylated ubiquitin have not been assessed.…”

mentioning

confidence: 99%

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Shang

Deng

Liu

et al. 2005

Journal of Biological Chemistry

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“…It is possible that the association of ␣-and ␤-Spectrin blocks ubiquitination of ␣-Spectrin and its subsequent degradation via the proteasome. Ubiquitination has been previously reported for ␣-Spectrin (Corsi et al, 1995;Galluzzi et al, 2001) and may thus help to define the correct protein-expression levels.…”

Section: Discussionmentioning

confidence: 99%

Distinct functions of α-Spectrin and β-Spectrin during axonal pathfinding

Hülsmeier¹,

Pielage²,

Rickert

et al. 2007

Development

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Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect ␤-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slitsensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that ␤-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, ␤-Spectrin is associated with ␣-Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of ␤-Spectrin significantly reduces levels of neuronal ␣-Spectrin expression, whereas gain of ␤-Spectrin leads to an increase in ␣-Spectrin protein expression. Because the loss of ␣-spectrin does not result in an embryonic nervous system phenotype, ␤-Spectrin appears to act at least partially independent of ␣-Spectrin to control axonal patterning.

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“…Recent observations suggest that fodrin, an analog of spectrin, which is a major component of the cortical cytoskeleton of most eukaryotic cells, is degraded during apoptosis in a variety of cells, including T-lymphoma cells [19]. Moreover, it is known that c~-spectrin can be ubiquitinated [20], and that cells deficient in spectrin tend to show a blebbing reminiscent of the morphology of apoptotic cells [21]. In addition, partial degradation of actin during apoptosis was observed recently in human leukemia U93H cells [22].…”

Section: F Pitzer Et Al/febs Letters 394 (1996) 47 50mentioning

confidence: 99%

Removal of proteasomes from the nucleus and their accumulation in apoptotic blebs during programmed cell death

et al. 1996

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Apoptosis can be initiated in immortalized cAMPstimulated rat ovarian granulosa cells by induction of wild-type p53 activity, lmmunocytochemical studies using confocal microscopy reveal that in apoptotie, unlike in normal growing cells, the proteasomes are removed from the nucleus and accumulate within the apoptotic blebs at the periphery of the cell. In parallel, a striking reorganization of the actin cytoskeleton is observed which forms a spherical network separating the apoptotic blebs from the cytoplasmic organelles, such as mitochondria and lipid droplets which remain in the perinuclear region. The reorganization of the actin cytoskeleton as well as disappearance of proteasomes from the nucleus suggest possible function of proteasomes in apoptotic regulation. [16]. In such cells, shifting the temperature of growth from 37°C to 32°C allows the manifestation of the wild-type p53 phenotype, as evident by up-regulation of the WAF-1/CIP-1 gene and arrest of cell growth.We found that while most of the proteasomes were accumulated in the apoptotic blebs during programmed cell death, the organelles such as mitochondria and lipid droplets remain intact in the perinucomiclear region during this process. Interestingly, confocal microscopy shows that concomitant with the sequestering of the proteasomes, the cytoskeleton is rearranged creating a barrier which separates organelles from the apoptotic blebs.

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Ubiquitin Is Conjugated to the Cytoskeletal Protein α-Spectrin in Mature Erythrocytes

Cited by 32 publications

References 36 publications

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Distinct functions of α-Spectrin and β-Spectrin during axonal pathfinding

Removal of proteasomes from the nucleus and their accumulation in apoptotic blebs during programmed cell death

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